In the kidney, the majority of volume is reabsorbed in the proximal tubule (PT). The maintenance of unidirectional transepithelial Na transport in PT requires that K brought into the cell across the basolateral membrane (BLM) by the Na,K-ATPase pump be able to recycle back across the BLM through a K channel. The concept of "pump-leak coupling" refers to the increase in BLM K conductance (GK) that occurs in association with increased activity of the Na,K-ATPase pump. Indeed, a major question in renal physiology is understanding exactly how an increase in transepithelial transport leads to an increase in the dominant BLM K conductance. Despite its pivotal physiological role, relatively little is known about the in situ regulation of the dominant BLM K channel, and its molecular identity remains unknown. Here we plan to use a combination of electrophysiology and molecular biology to determine the molecular physiology and identity of this K channel in amphibian and mammalian PT. In Aim 1, single and ensemble K currents will be characterized using novel macropatch clamp recording from intact mouse PT testing the hypothesis that it is an ATP-sensitive K channel. Aim 2 explores the in situ behavior of the channel during modulation of transepithelial transport by major signaling pathways in the PT. We hypothesize that changes in transcellular Na flux, angiotensin II (Ang II), and arachidonic acid (AA) are critical regulators of transport, and therefore the K channel, in the PT. Aim 3 proposes to establish the molecular identity of this K channel using homology cloning combined with functional expression and antisense nucleotides in native salamander PT cells. The recombinant channel, which is hypothesized to be a heteromultimer comprised of a Kir6.1 subunit and a SUR2B sulfonylurea receptor, will be expressed and compared to the native channel in amphibian and mammalian PT. Success in these aims will deepen our understanding of salt and water handling in the PT and pave the way for subsequent structure-function studies with mutant channels. This work may also have important clinical implications, as therapeutic agents that interfere with the Ang II system (e.g., ACEIs, A2RBs) and AA (e.g., NSAIDS), which are among the most commonly prescribed medications. Therefore, new knowledge gained from the proposed studies are important in health, and will be highly relevant to the pathophysiology of hypertension, dysregulation of extracellular volume, and renal insufficiency. [unreadable] [unreadable]